The exocrine pancreas and intestine play essential roles in vertebrate physiology. For both organs cell renewal also has an important homeostatic role. In the intestine, cells of the villus epithelium are continuously replaced by the differentiated progeny of stem cell progenitors that reside within the crypts surrounding each villus. In the pancreas, multipotent progenitors reside within the ductular epithelium and believed to serve as a source of differentiated endocrine and acinar cells that can replace cells lost under specific experimental and pathological conditions. Identification of the genes that regulate cell lineage specification and differentiation of digestive organ stem cell progenitors is important to research related to common human diseases such as cancers of the pancreas and colon. Genetic analysis is an efficient means to identify genes that regulate developmental processes, particularly organogenesis. In this application we outline a strategy for the molecular characterization of lineage specification and cell differentiation in the developing exocrine pancreas and intestine of the zebrafish, a vertebrate model system uniquely suited to this purpose. The exocrine pancreas and intestine of zebrafish larvae function at five days post&#8209;fertilization, a stage amenable to standard mutagenesis techniques. Further, progenitors and differentiated cells of both organs are easily visualized throughout development using organ specific markers. The first Aim of this application is to perform a systematic mutagenesis screen for genes that regulate lineage specification and cell differentiation during exocrine pancreas and intestinal development. Using in situ and histochemical analysis we will identify genes that regulate growth and differentiation of the exocrine pancreas as well as development of the goblet and enteroendocrine cell lineages of the intestine. The feasibility of such as screen was proven in a recent pilot study. The second Aim is to identify molecular markers in the exocrine pancreas and intestine of larval zebrafjsh by generating a zebrafish pancreas and intestine EST database. This aim will be performed in collaboration with members of the zebrafish genome community. The third Aim is to map the mutations and ESTs using meiotic and physical mapping techniques. This aim will allow identification of candidate genes for the mutations recovered in Specific Aim 1, and provide a databank of targets for gene knockdown experiments. The fourth aim of this application is to explore the function of novel genes identified in Specific Aim 2 using a morpholino based gene knockdown strategy.